Exp. Anim. 60(1), 65–70, 2011
Triplex PCR for the Simultaneous Detection of Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium Eui-Suk JEONG1), Kyoung-Sun LEE1), Seung-Ho HEO1, 2), Jin-Hee SEO1), and Yang-Kyu CHOI1) 1)
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, Seoul 143-701 and 2)Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea
Abstract: The accurate and economical diagnosis of pathogenic bacteria is necessary for the microbiological control of laboratory animals. In this study, we developed a triplex PCR method for the direct detection of three common gastroenteric bacteria, Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium. Targets were specifically amplified by conventional PCR assay using a genomic fragment from P. aeruginosa, 16S ribosomal RNA from H. hepaticus, and the invA gene from S. typhimurium. To investigate the specificity of our primers, they were tested against purified DNA from many other bacterial species. There were no amplification products from other bacteria. Under optimized conditions, the triplex assay simultaneously yielded a 726-bp product from P. aeruginosa, a 417-bp product from H. hepaticus, and a 246-bp product from S. typhimurium. The detection limits of this assay in pure culture were 10 pg for P. aeruginosa, and 0.1 pg for H. hepaticus and S. typhimurium. All three bacteria were successfully detected in the liver, cecum, and feces of experimentally infected mice. This method is a useful and convenient assay that allows the simultaneous identification of bacterial pathogens in mice. Our triplex method will be used to improve quality control in the detection of pathogenic bacterial infections in laboratory animal facilities. Key words: H. hepaticus, P. aeruginosa, S. typhimurium, triplex PCR
Introduction Pseudomona aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium are responsible for gastroenteric diseases in laboratory animals . P. aeruginosa is widespread in nature, inhabiting soil, water, plants, and animals, and is as an opportunistic pathogen of mice. P. aeruginosa is transmitted via contact with contami-
nated water, feed, bedding, and infected rodents and humans . P. aeruginosa infection in mice and rats could affect a variety of research projects, depending upon the organ systems affected  and was found to be a major gastroenteric bacterium in mouse and rat facilities in Korea . The genus Helicobacter naturally infects mice and rats and consists of H. hepaticus, H. bilis, H. muridarum, H. trogontum, and H. rodentium
(Received 19 July 2010 / Accepted 19 September 2010) Address corresponding: Y.-K. Choi, Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, #1 Hwayangdong, Gwangjin-gu, Seoul 143-701, Republic of Korea
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[2, 15]. Among these, H. hepaticus is the most pathogenic cause of infection in mice. The prevalence of H. hepaticus is currently unknown but is suspected to be quite high . H. hepaticus was first discovered in association with hepatitis, hepatocellular adenoma, and adenocarcinoma in some strains of mice. Clinical signs of H. hepaticus infection are absent in immunocompetent mice but can include rectal prolapse in immunodeficient mice . Salmonella spp. are found as pathogens in immunocompromised mice and rats. S. typhimurium is a zoonotic agent and the most common serotype infecting laboratory rodents. Although the prevalence of asymptomatic carriers is unknown, it is probably low. S. typhimurium is the most frequently identified serovar of Salmonella in Italy . Recently, S. typhimurium was isolated from frozen vacuum-packed rodents in Texas in the USA . As a source of Salmonella, rodent facilities need to be evaluated to protect human and animal health. Transmission of S. typhimurium is caused by ingestion of contaminated feed ingredients or water, and by contact with contaminated bedding and animal facility personnel . We selected three bacteria in this study, P. aeruginosa, H. hepaticus, and S. typhimurium, based on their prevalence or zoonotic character among gastroenteric bacteria. Microbiological culture is the principal tool used to diagnose infectious diseases. Commonly, microbial contamination is assessed by culture methods. Most pathogenic bacteria are easily grown on culture agar. However, it is not easy to identify bacteria, especially at the species level by culture-based methods. PCR assays are useful and convenient methods for the rapid identification of bacterial species. The accurate and economical diagnosis of pathogenic bacteria is necessary for microbiological control of laboratory animals. PCR assays provide a useful and convenient method for the rapid identification of bacterial pathogens in laboratory animals. To rapidly and economically diagnose bacteria commonly found in mice, we designed specific primer sequences targeting three gastroenteric bacteria, P. aeruginosa, H. hepaticus, and S. typhimurium, using NCBI BLAST. The target nucleic acid fragments were specifically amplified by conventional PCR. This allowed us to develop a triplex PCR assay for simultaneous detection of P. aeruginosa, H. hepaticus, and S. typhimurium.
Materials and Methods Bacterial strains The sixteen reference strains that were used in this study included six P. aeruginosa strains, ATTC 10145 (type strain), KUV 101, KUV 102, KUV 103, KUV 104, and KUV 105; two S. typhimurium strains, ATCC 13311 (type strain) and M15; one CAR bacillus strain; one Corynebacterium kutscheri strain, ATCC 15677 (type strain); one H. hepaticus strain ATCC 51448 (type strain); one Klebsiella pneumoniae strain ATCC 13883 (type strain); one Mycoplasma pulmonis strain ATCC 19612 (type strain); one Pasteurella multocida strain ATCC 43137 (type strain); one Pasteurella pneumotropica strain ATCC 35149 (type strain); and Streptococcus pneumoniae ATCC 33400 (type strain). The type strains of P. aeruginosa, S. typhimurium, Helicobacter hepaticus, Klebsiella pneumoniae and Pasteurella pneumotropica were provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and the Korean Collection for Type Culture. The type strains of Corynebacterium kutscheri, Mycoplasma pulmonis, Pasteurella multocida, and Streptococcus pneumoniae were obtained from the American Type Culture Collection (ATCC). And DNA of Clostridium piliforme (strain RJ), Citrobacter rodentium (ATCC 51459), and Helicobacter bilis (ATCC 51630) were provided by KRIBB. P. aeruginosa was grown on cetrimid agar (Merck, Darmstadt, Germany) at 37°C for 24 h. H. hepaticus was grown on 5% sheep blood agar at 37°C for 72–96 h under the microaerobic conditions of 5% CO2, 5% O2, and 90% N2. S. typhimurium was grown on DHL agar (Merck) at 37°C for 48 h. Corynebacterium kutscheri, Klebsiella pneumoniae, Pasteurella multocida, Pasteurella pneumotropica, and Streptococcus pneumoniae were grown on 5% sheep blood agar at 37°C for 24–48 h. Mycoplasma pulmonis was grown on mycoplasma agar (Oxoid, Hampshire, UK) combined with mycoplasma selective supplement-G (Oxoid) at 37°C for 7 days. To determine the concentrations of bacteria, P. aeruginosa and S. typhimurium were grown overnight in brain heart infusion broth at 37°C with agitation, H. hepaticus was grown in brain heart infusion broth, consisting of 10% FBS supplemented with 10 mg/l vancomycin and 2.5 mg/l amphotericin B under microaerobic conditions.
TRIPLEX PCR OF ENTERIC BACTERIA Table 1. Primers used in this study Species P. aeruginosa H. hepaticus
Sequences (5’ to 3’)
Product size (bp)
TAT TTC AAG GAT GGC TCC AC GCG TTG GTT GTC CAA GTT TA
In this study
Shames et al. (1995) 
Kim et al. (2005) 
GCA TTT GAA ACT GTT ACT CTG CTG TTT TCA AGC TCC CC
ATT AAT TAT GGA AGC GCT CGC ATT GTA ATG AGA TCC ATC AAA TTA GCG
The bacteria were pelleted by centrifuging at 4,000 rpm for 5 min and washed three times in sterile PBS. The concentration of bacteria was determined by measuring the absorbance values at 600 nm and then plotting the optical density on a standard curve generated from known CFU (colony forming units) values. The number of CFU/ml was determined by plating serial dilutions. Preparation of DNA Samples Two methods were used for DNA extraction. First, template DNA was isolated from bacterial culture by modifying the boiling method. Briefly, a 1-ml aliquot of each bacterial culture was centrifuged at 10,000 rpm at 4°C for 5 min. The supernatant was carefully removed, and the cell pellet was washed in 1 ml PBS and then mixed with 100 µl of PBS and 1% Triton ×100. The tube was incubated for 10 min at 95°C in a water bath and immediately chilled on ice for 5 min. After centrifugation at 10,000 rpm at 4°C for 5 min, the supernatant containing DNA was carefully transferred to a new tube. A 1-µl aliquot was used as template DNA for the PCR. All DNA preparations were stored at –20°C until use. Second, template DNA was extracted from tissue and feces using an AccuPrep Genomic DNA Extraction Kit (Bioneer Inc., Daejeon, Korea). Briefly, 500 µl of a lysis solution was added to each microtube and incubated at 60°C overnight using a water-bath. Following incubation, 100 µl of isopropanol was added, and mixed well by pipetting. The lysate was carefully transferred into the upper reservoir of the binding column tube without wetting the rim, and then centrifuged at 8,000 rpm for 1 min. Five hundred microliters of washing solution (I, II) were added, and the samples were centrifuged at 12,000 rpm for 1 min. The binding column was transferred to a new 1.5 ml tube for elution, then 100 μl of elution buffer was added and allowed to sit for least 1
min at 15–25°C. Finally, samples were centrifuged at 8,000 rpm for 1 min for elution. The concentration of DNA used was 100 ng/µl. A 1-µl aliquot was used as template DNA for the PCR. All DNA preparations were stored at –20°C until use. PCR amplification DNA was amplified in a 20 µl PCR mixture containing 30 mM KCl, 10 mM Tris (pH 9.0), 1.5 mM MgCl2, 250 µM of each deoxynucleoside triphosphate (dTTP, dATP, dCTP, and dGTP), 10 pmol of each primer, template DNA, and 1 unit of Taq DNA polymerase (Bioneer Inc.). The DNA primers used in this study are listed in Table 1. The cycling conditions were: initial denaturation for 5 min at 95°C, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, followed by an additional 10 min at 72°C for final extension. After the reaction, PCR amplified products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Experimental infection with P. aeruginosa, H. hepaticus, or S. typhimurium Specific pathogen-free BALB/c mice were purchased at 6 to 7 weeks of age from KRIBB. Mice were maintained in a pathogen-free room on a 12:12 light cycle. To determine whether BALB/c mice were free of P. aeruginosa, H. hepaticus, and S. typhimurium or not, we used our triplex PCR and conventional culture methods. We didn’t detect any bacteria in the BALB/c mice. For mouse inoculation, type strain cultures of P. aeruginosa, H. hepaticus, and S. typhimurium were adjusted to 107–9 CFU/200 µl. Three mice were infected with 200 µl of one of the cultures by intragastric gavage and the actual dose of bacteria used was confirmed by plating on agar plates. Once infection was established (1 to 8
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Fig. 1. Specificity of the triplex PCR for detection of Pseudomonas aeruginosa (726 bp), Helicobacter hepaticus (417 bp), and Salmonella enterica serovas typhimurium (246 bp). M, 100 bp ladder size marker; lane 1, negative control (no template); lane 2, CAR bacillus; lane 3, Corynebacterium kutscheri; lane 4, H. hepaticus; lane 5, Klebsiella pneumoniae; lane 6, Mycoplasma pulmonis; lane 7, Pasteurella multocida; lane 8, Pasteurella pneumotropica; lane 9, P. aeruginosa; lane 10, S. typhimurium; lane 11, Streptococcus pneumoniae; lane 12, P. aeruginosa KUV 101; lane 13, P. aeruginosa KUV 102; lane 14, P. aeruginosa KUV 103; lane 15, P. aeruginosa KUV 104; lane 16, P. aeruginosa KUV 105; lane 17, S. typhimurium M15; lane 18, mixture of P. aeruginosa, H. hepaticus, and S. typhimurium single colony. PCR assays were done with template concentrations of up to 100 ng.
weeks after inoculation), and confirmed by fecal PCR, the mice were sacrificed. Liver, cecum, and feces were collected to evaluate the triplex PCR system. All procedures were approved by the Institutional Animal Care and Use Committee of Konkuk University. Results Specificity of triplex PCR The tiplex PCR primers were designed to target DNA specific to P. aeruginosa, H. hepaticus, and S. typhimurium, as described in Table 1, which includes the primer sequences and products size. To investigate the specificity of the selected P. aeruginosa, H. hepaticus, and S. typhimurium primers, each was tested individually and in combination. The primer pairs yielded amplified PCR products of 726, 417, and 246 bp for P. aeruginosa, H. hepaticus, and S. typhimurium, respectively (Fig. 1). DNA was isolated from 15 bacterial strains that can be found in the gastrointestinal tract and used to test the specificity of this assay. None of the primer pairs produced any amplicons of CAR bacillus, C. kutscheri, K.
Fig. 2. Sensitivity of the single and triplex PCRs for detection of Pseudomonas aeruginosa (726 bp), Helicobacter hepaticus (417 bp), and Salmonella enterica serovas typhimurium (246 bp). (A) M, 100 bp ladder size marker; lane 1, negative control (no template); lane 2, 1 ng; lane 3, 100 pg; lane 4, 10 pg; lane 5, 1 pg; lane 6, 0.1 pg concentration of P. aeruginosa, H. hepaticus, and S. typhimurium. (B) M, 100 bp ladder size marker; lane 1, negative control (no template); lane 2, P. aeruginosa (100 ng), H. hepaticus (0.1 pg) and S. typhimurium (0.1 pg); lane 3, P. aeruginosa (10 ng), H. hepaticus (1 pg), and S. typhimurium (1 pg); lane 4, P. aeruginosa (1 ng), H. hepaticus (10 pg), and S. typhimurium (10 pg); lane 5, P. aeruginosa (100 pg), H. hepaticus (100 pg), and S. typhimurium (100 pg); lane 6, P. aeruginosa (10 pg), H. hepaticus (1 ng), and S. typhimurium (1 ng); lane 7, P. aeruginosa (1 pg), H. hepaticus (10 ng), and S. typhimurium (10 ng); lane 8, P. aeruginosa (0.1 pg), H. hepaticus (100 ng), and S. typhimurium (100 ng).
pneumoniae, M. pulmonis, P. multocida, P. pneumotropica, or S. pneumoniae. The P. aeruginosa primer pair specifically amplified the expected 726 bp product from all P. aeruginosa strains, KUV 101 to KUV 105. Also, the primer pairs did not produce any amplicons from DNA of Clostridium piliforme, Citrobacter rodentium, and Helicobacter bilis (data not shown). These results demonstrate there was no cross reactivity in our assay. Sensitivity of the single and triplex PCRs The sensitivity of the single and triplex PCR methods was assessed by serial 10-fold dilutions of DNA isolated from pure cultures of the type strains of P. aeruginosa, H. hepaticus, and S. typhimurium. Single PCR was able to detect 0.1 pg of P. aeruginosa, H. hepaticus, and S. typhimurium DNA (Fig. 2A). Our triplex method was able to detect P. aeruginosa at 10 pg concentration, and H. hepaticus and S. typhimurium at 0.1 pg concentrations (Fig. 2B).
TRIPLEX PCR OF ENTERIC BACTERIA
Fig. 3. Triplex PCR detection of Pseudomonas aeruginosa (726 bp), Helicobacter hepaticus (417 bp), and Salmonella enterica serovas typhimurium (246 bp) in cecum, liver, and feces. M, 100 bp ladder size marker; lane 1, negative control (no template); lanes 2–4, mice infected by P. aeruginosa; lanes 5–7, mice infected by H. hepaticus; lanes 8–10, mice infected by S. typhimurium.
Evaluation of the triplex PCR in experimentally infected mice Triplex PCR was evaluated in vivo using feces, cecum, and livers collected from experimentally infected mice. Amplification with P. aeruginosa specific primers yielded a PCR product of 726 bp from the murine cecum and feces. H. hepaticus specific primers yielded an amplified PCR product of 417 bp in the murine liver, cecum, and feces. S. typhimurium specific primers yielded an amplified PCR product of 246 bp in the murine liver, cecum, and feces (Fig. 3). Discussion We have described the simultaneous detection of the three mouse gastroenteric pathogens, P. aeruginosa, H. hepaticus, and S. typhimurium. These bacteria were selected because of their prevalence in Korea  or their zoonotic character  from among gastroenteric bacteria. In multiplex PCR, two or more primer pairs are included in one reaction tube and two or more DNA templates are targeted simultaneously. This is a relatively simple molecular way to detect multiple bacterial strains in one PCR. In multiplex PCR, primer pairs need to be specific to the genes of interest and the PCR products should be of different sizes . In this study, target nucleic acid fragments were specifically amplified by conventional PCR of the 16S ribosomal RNA of H. hepaticus, a genomic fragment from P. aeruginosa, and the
invA gene of S. typhimurium. There was no cross amplification of the three targets with other murine bacteria, including CAR bacillus, C. kutscheri, K. pneumoniae, M. pulmonis, P. multocida, P. pneumotropica, and S. pneumoniae. Multiplex PCR for Escherichia coli O157:H7, Salmonella Typhimurium, and Shigella ﬂexneri has been performed in apple cider , and the simultaneous detection of Salmonella enteritidis, typhi, and typhimurium has been reported in poultry meat [1, 9]. However, to the best of our knowledge, the simultaneous detection of H. hepaticus, P. aeruginosa, and S. typhimurium has not previously been performed. In this study, the detection limits of the triplex PCR assay ranged from 0.1 to 10 pg of DNA, and it was able to detect the mixture of P. aeruginosa, H. hepaticus, and S. typhimurium from a single colony on an agar plate (data not shown). However, the sensitivity of the triplex PCR method was less than those of the single PCRs (Fig. 2). Yuan et al. reported that 0.5 pg DNA from Escherichia coli, Listeria monocytogenes, and Salmonella spp. was sufficient for detection by a triplex PCR method . The level of sensitivity described by them suggests that the triplex PCR assay described in this study will be useful for the detection of gastroenteric diseases in rodents. We examined the applicability of our PCR assay by testing organs from infected mice, including feces, cecum, and liver. The three bacteria were successfully identified in mice using our triplex PCR assay (Fig. 3). Recently, after toxigenic strains of P. aeruginosa (PA 103) were inoculated subcutaneously into a burned area, P. aeruginosa were isolated from sera, spleen, and liver . However, we did not detect P. aeruginosa in liver samples. This may be due to differences in the bacterial strain and/or infection route. The triplex PCR is suitable for diagnosis of P. aeruginosa, H. hepaticus, and S. typhimurium infection in clinical cases. In conclusion, our PCR assay is simple, rapid, sensitive, and specific, and allows the simultaneous detection of P. aeruginosa, H. hepaticus, and S. typhimurium. This assay allows detection directly from bacterial colonies, and from tissues of experimentally infected mice. The speed and ease of our PCR method also makes it convenient for use in clinical laboratories for the diagnosis of gastroenteric bacterial infections. Our method can be
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used to detect and subsequently eradicate pathogenic bacterial infection from laboratory mice facilities, and allows for quality control of laboratory animal populations. Further, our assay will facilitate the epidemiological study of infectious disease outbreaks in mice. Acknowledgments This study was funded by a Korea Food and Drug Administration grant (08152 kitasa 628). We thank Dr. Won YS at the Korea Research Institute of Bioscience and Biotechnology for sending the DNA of several bacteria. References 1. de Freitas, C.G., Santana, A.P., da Silva, P.H., Gonçalves, V.S., Barros, M. de A., Torres, F.A., Murata, L.S., and Perecmanis, S. 2010. PCR multiplex for detection of Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat. Int. J. Food Microbiol. 139: 15–22. 2. Fox, J.G. and Lee, A. 1997. The role of Helicobacter species in newly recognized gastrointestinal tract diseases of animals. Lab. Anim. Sci. 47: 222–255. 3. Kim, S.H., Kim, S.H., Lee, S.W., Kang, Y.H., and Lee, B.K. 2005. Rapid serological identification for monophasic Salmonella serovars with a hin gene-specific polymerase chain reaction. J. Bacteriol. Virol. 35: 291–297. 4. Lee, K.M., McReynolds, J.L., Fuller, C.C., Jones, B., Herrman, T.J., Byrd, J.A., and Runyon, M. 2008. Investigation and characterization of the frozen feeder rodent industry in Texas following a multi-state Salmonella Typhimurium outbreak associated with frozen vacuum-packed rodents. Zoonoses Public Health 55: 488–496. 5. Li, Y. and Mustapha, A. 2004. Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR. J. Food Prot. 67: 27–33. 6. Manafi, A., Kohanteb, J., Mehrabani, D., Japoni, A., Amini, M., Naghmachi, M., Zaghi, A.H., and Khalili, N. 2009. Active immunization using exotoxin A confers protection against Pseudomonas aeruginosa infection in a mouse burn model. BMC Microbiol. 9: 23.
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